Structure of LRRK2 in Parkinson's disease and model for microtubule interaction.

Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson's disease1 and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3 and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson's disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

Dynamics of antibiotic resistance markers and Escherichia coli invasion in riverine heterotrophic biofilms facing increasing heat and flow stagnation.

As motivation to address environmental dissemination of antimicrobial resistance (AMR) is mounting, there is a need to characterize mechanisms by which AMR can propagate under environmental conditions. Here we investigated the effect of temperature and stagnation on the persistence of wastewater-associated antibiotic resistance markers in riverine biofilms and the invasion success of genetically-tagged Escherichia coli. Biofilms grown on glass slides incubated in-situ downstream of a wastewater treatment plant effluent discharge point were transferred to laboratory-scale flumes fed with filtered river water under potentially stressful temperature and flow conditions: recirculation flow at 20 °C, stagnation at 20 °C, and stagnation at 30 °C. After 14 days, quantitative PCR and amplicon sequencing were used to quantify bacteria, biofilms diversity, resistance markers (sul1, sul2, ermB, tetW, tetM, tetB, blaCTX-M-1, intI1) and E. coli. Resistance markers significantly decreased over time regardless of the treatment applied. Although invading E. coli were initially able to colonize the biofilms, its abundance subsequently declined. Stagnation was associated with a shift in biofilm taxonomic composition, but there was no apparent effect of flow conditions or the simulated river-pool warming (30 °C) on AMR persistence or invasion success of E. coli. Results however indicated that antibiotic resistance markers in the riverine biofilms decreased under the experimental conditions in the absence of exposure to external inputs of antibiotics and AMR.

Effects of neo-adjuvant chemotherapy for oesophago-gastric cancer on neuro-muscular gastric function.

Delayed gastric emptying symptoms are often reported after chemotherapy. This study aims to characterise the effects of chemotherapy on gastric neuro-muscular function. Patients undergoing elective surgery for oesophago-gastric cancer were recruited. Acetylcholinesterase, nNOS, ghrelin receptor and motilin expressions were studied in gastric sections from patients receiving no chemotherapy (n = 3) or oesophageal (n = 2) or gastric (n = 2) chemotherapy. A scoring system quantified staining intensity (0-3; no staining to strong). Stomach sections were separately suspended in tissue baths for electrical field stimulation (EFS) and exposure to erythromycin or carbachol; three patients had no chemotherapy; four completed cisplatin-based chemotherapy within 6 weeks prior to surgery. AChE expression was markedly decreased after chemotherapy (scores 2.3 ± 0.7, 0.5 ± 0.2 and 0 ± 0 in non-chemotherapy, oesophageal- and gastric-chemotherapy groups (p < 0.03 each) respectively. Ghrelin receptor and motilin expression tended to increase (ghrelin: 0.7 ± 0.4 vs 2.0 ± 0.4 and 1.2 ± 0.2 respectively; p = 0.04 and p = 0.2; motilin: 0.7 ± 0.5 vs 2.2 ± 0.5 and 2.0 ± 0.7; p = 0.06 and p = 0.16). Maximal contraction to carbachol was 3.7 ± 0.7 g and 1.9 ± 0.8 g (longitudinal muscle) and 3.4 ± 0.4 g and 1.6 ± 0.6 (circular) in non-chemotherapy and chemotherapy tissues respectively (p < 0.05 each). There were loss of AChE and reduction in contractility to carbachol. The tendency for ghrelin receptors to increase suggests an attempt to upregulate compensating systems. Our study offers a mechanism by which chemotherapy markedly alters neuro-muscular gastric function.

Clinicopathologic Correlation of Lens Epithelial Metaplasia and Late Intraocular Lens Dislocation After Repair of Retinal Detachment.

IMPORTANCE: In-the-bag intraocular lens dislocation is an uncommon but serious complication of cataract surgery in patients with previous repair of retinal detachment. The causative mechanism is currently unknown. We report histologic findings from a retrospective case series from 1993 to 2010 and suggest a possible mechanism to explain this association. OBSERVATIONS: Clinical characteristics of 8 patients presenting with in-the-bag intraocular lens dislocation after repair of retinal detachment were evaluated. Explanted capsular bags from 3 of these patients were compared with pathologic changes of crystalline lenses associated with retinal detachment. Histologic examination of the explanted capsular bags revealed a paucicellular membrane that covered the concertina-like folded surface of the lens capsule. The lens capsule was devoid of epithelial cell nuclei and showed excessive thickening with the presence of spindle-shaped cells, such as fibroblasts. Collagen fibers were noted in the extracellular matrix. CONCLUSIONS AND RELEVANCE: Previous studies of crystalline lens pathologic findings associated with retinal detachment have shown changes in the epithelium with migration and subsequent metaplasia of epithelial cells, resulting in excessive thickening of the anterior capsule with a layer of fibrous tissue. In this retrospective series, similar histologic findings were seen, suggesting that zonular dehiscence and lens dislocation may result from progressive capsular contraction secondary to retinal detachment-induced lens epithelial metaplasia.

Additive effects of weight-bearing exercise and estrogen on bone mineral density in older women.

The separate and combined effects of weight-bearing exercise and hormone replacement therapy (HRT) on bone mineral density (BMD) were studied in 32 women, 60 to 72 years of age. HRT consisted of continuous conjugated estrogens 0.625 mg/day and trimonthly medroxyprogesterone acetate 5 mg/day for 13 days. Exercise consisted of 2 months of low-intensity exercise followed by 9 months of more vigorous weight-bearing exercise approximately 45 minutes/day, > or = 3 days/week, at 65-85% of maximal heart rate. Lumbar spine and proximal femur BMD were significantly increased in response to exercise and to HRT, and total body BMD was significantly increased in response to HRT; neither exercise nor HRT had an effect on wrist BMD. The combination of exercise + HRT resulted in increased BMD at all sites except the wrist, with effects being additive for the lumbar spine and Ward's triangle and synergistic for the total body. Based on reductions in serum osteocalcin levels, it appears that increases in BMD in response to HRT and exercise + HRT were due to decreased bone turnover. The lack of change in serum osteocalcin and IGF-I in response to exercise alone suggests that increases in BMD were due to decreased bone resorption and not increased formation. Results indicate that weight-bearing exercise + HRT may be effective in preventing and/or treating osteoporosis. It is likely that the additive effects of weight-bearing exercise and HRT on bone mineral accretion, coupled with other adaptations to the exercise (i.e., increased strength and functional capacity), could effectively reduce the incidence of falls and osteoporotic fractures.

Dietary patterns, eating behaviors, and bone mineral density in women runners.

Although reduced gonadal steroid hormone concentrations appear to play a major role in lower trabecular bone mineral density (BMD) in women with athletic amenorrhea, dietary deficiencies and eating behaviors may also affect BMD in women runners. To investigate this possibility, dietary patterns (7-d records), eating-disorders inventory (EDI), and BMD were examined in nine nonrunning eumenorrheic control (Contl) and 32 women runners classified as eumenorrheic (n = 19, Eumen) and oligo/amenorrheic (a group in which some were oligomenorrheic and some were amenorrheic; Ol/Am, n = 13). Runner groups had similar cardiorespiratory fitness, body composition, and training characteristics. Lumbar spine BMD was lower in the Ol/Am runners (-12%, P less than 0.05) but proximal femur BMD did not differ. Dietary intake and EDI subscale scores were similar among the groups. However, there was an inverse trend between EDI subscale scores for bulimia and ineffectiveness and femoral BMD in the Ol/Am runners (r = -0.62 to -0.71, P less than 0.05). These results suggest that self-reported dietary intake and/or eating behaviors do not predict reproductive-function alterations in women runners, but eating behaviors may be associated with lower BMD in Ol/Am runners.

A new immunohistochemical antibody for the assessment of estrogen receptor status on routine formalin-fixed tissue samples.

We describe a method for the immunocytochemical assessment of estrogen receptor (ER) status on routinely processed, formalin-fixed tissue using a recently developed commercially available monoclonal antibody (Dako 1D5) with a microwave antigen retrieval technique. A series of 90 cases of human breast carcinoma was analyzed and the staining was assessed using a semiquantitative microscopic scoring method and by assessment of the percentage of nuclei showing positive staining. The results were compared with assessment using another commercially available antibody (Abbott H222) and with clinical response to tamoxifen therapy. Direct comparison of the paired sets of H scores and the percentage of positively stained nuclei using the two different techniques showed a significant correlation. By assigning an arbitrary cut-off for positivity of H score = 50, assessment of ER status using DAKO 1D5 antibody was found to correlate with response to tamoxifen therapy with a sensitivity of 90% and a specificity of 51%, similar to values in previous studies using other methods. We conclude that assessment of ER status using this new antibody and technique gives accurate results on routinely processed, formalin-fixed tissue and may be used as an alternative to other methods.

Age-related differences in body composition by hydrodensitometry and dual-energy X-ray absorptiometry.

To determine whether percent body fat (%BF) is overestimated in older people by hydrodensitometry (HD) because of an age-related decrease in bone mineral content (BMC), body composition of 113 women and 72 men (21-81 yr) was assessed by HD and dual-energy X-ray absorptiometry (DEXA). DEXA provides an estimate of %BF adjusted for differences in BMC. HD %BF and DEXA %BF were not different in young people [21-39 yr; 17.6 +/- 6.4 (SD) vs. 17.6 +/- 7.2%, NS], were slightly, but significantly, different in middle-aged people (40-59 yr; 25.5 +/- 6.4 vs. 24.1 +/- 6.7%, P < 0.05), and showed the largest disparity in older people (> or = 60 yr; 34.9 +/- 7.9 vs. 30.8 +/- 8.7%, P < 0.05). The discrepancy in older people was apparently not due to mineral loss, however, inasmuch as correction of HD %BF for variance in BMC as a fraction of fat-free mass resulted in only small adjustments (approximately 1%) of %BF. Assessment of DEXA %BF was further evaluated in nine subjects with packets of lard (2-3 kg) overlying either the thigh or the trunk region. Only 55% of the exogenous fat was identified as fat when it was in the trunk region compared with 96% when it was positioned over the legs. These data suggest that the age-related increase in upper body adipose tissue is underestimated by DEXA.

Release of luteinizing hormone and growth hormone after recovery from maximal exercise.

Pulsatile properties of luteinizing hormone (LH) and growth hormone (GH) release were evaluated in 19 eumenorrheic untrained females [mean age 31.1 +/- 1.1 yr, height 165.2 +/- 1.4 cm, weight 64.8 +/- 2.1 kg, peak oxygen uptake (Vo2) 41.6 +/- 1.4 (SE) ml.kg-1.min-1] during the early follicular phase of the menstrual cycle (days 3-4 after the onset of menses). Each subject was studied during two consecutive menstrual cycles under each of two conditions in random order: 1) no formal exercise for 72 h (C) and 2) 12-24 h after two maximal exercise bouts (peak Vo2/lactate threshold treadmill evaluation and a 3,200-m time-trial run or a maximal Vo2 inclined treadmill test) performed on consecutive days (EX). Blood sampling was performed every 10 min for 12 h. LH and GH pulsatile parameters were identified and characterized by the Cluster pulse detection algorithm. No significant differences were noted in the number of peaks, peak amplitude, interpeak interval, peak increment, or 12-h integrated concentrations between C and EX for LH or GH. We conclude that maximal exercise protocols typically used for exercise evaluation do not have an effect on the pulsatile characteristics of LH or GH release in untrained women during the early follicular phase of the menstrual cycle if 12-24 h of recovery are allowed before evaluation of the pulsatile secretion of gonadotropins or GH.

Reproductive hormones and bone mineral density in women runners.

We examined the relationships among reproductive hormone concentrations and bone mineral density (BMD) in 43 women runners classified as eumenorrheic (n = 24), oligomenorrheic (n = 8), or amenorrheic (n = 11). Results were compared with a eumenorrheic nonrunner control group (n = 11). Serum 17 beta-estradiol, progesterone, and dehydroepiandrosterone sulfate concentrations were determined in daily blood samples for 21 days, and integrated concentrations (areas under the curve) were calculated. BMD was assessed at the lumbar spine and proximal femur by dual-photon absorptiometry. As expected, 17 beta-estradiol, progesterone, and lumbar spine BMD were higher in the control and eumenorrheic runner groups than in the oligomenorrheic and amenorrheic runner groups (P less than 0.05). Progesterone concentration was significantly correlated with lumbar spine BMD in the eumenorrheic runners (r = 0.61). None of the steroid hormones was significantly related to BMD in the oligomenorrheic/amenorrheic group. The present data suggest that circulating levels of gonadal steroid hormones affect axial BMD in eumenorrheic runners.

Durability of the reproductive axis in eumenorrheic women during 1 yr of endurance training.

Menstrual cycle (MC) alterations occur in some endurance-training women. We hypothesized that a prospective running program would evoke alterations in MC phase lengths and in the physiological frequency of pulses of luteinizing hormone (LH) and/or diminish 24-h integrated serum LH concentrations in some women. In addition, we postulated that women who train more intensively (above the lactate threshold) would show alterations in gonadotropin release earlier in the training program or to a greater degree. To test these hypotheses, we examined the effects of different exercise intensities on physiological and endocrine responses. Twenty-three healthy eumenorrheic gynecologically mature (postmenarchal age 17.8 +/- 0.9 yr) untrained women undertook a 1-yr training program at one of two exercise intensities, one at a velocity corresponding to the lactate threshold (LT) and the other halfway between that of LT and peak running velocity, or served as controls. Training distance was the same in each exercise group. Physiological measurements were repeated every four MC to track changes in fitness and readjust training velocities. The lengths of the MC and the follicular and luteal phases were determined from hormonal concentrations. Body composition, nutritional intake, and pulsatile release of LH were determined. The women ran approximately 790 miles. Each group improved physiologically, with the greater than LT group improving to a greater degree. A less than 2-day decrease in the luteal phase length was observed only in the greater than LT group. No significant changes for any parameter of pulsatile LH release were noted between exercise groups. No significant changes in nutritional intake and only small changes in body composition were noted in either exercise group despite the added energy expenditure of exercise. We conclude that a progressive exercise program of moderate distance and intensity does not adversely affect the robust reproductive system of gynecologically mature eumenorrheic women.

Identification of carcinoma cells in peripheral blood samples of patients with advanced breast carcinoma using RT-PCR amplification of CK7 and MUC1.

We have undertaken a pilot study to attempt to identify circulating carcinoma cells in a series of patients with advanced breast carcinoma, using reverse transcription-polymerase chain reaction (RT-PCR) to amplify mRNA of epithelial specific antigens. Using this method to amplify mRNA of MUC1 and cytokeratin 7 (CK7) the sensitivity of the technique was demonstrated by means of diluted concentrations of "spiked MCF7" cells in whole blood, showing a detection limit of 1 in 10(6) (CK7) and 1 in 10(5) (MUC1). Positive results were obtained from the peripheral blood of all nine female patients with advanced breast cancer for CK7 and eight of the nine patients for MUC1. CK7 was however detected in five of 11 healthy controls (eight females, three males) and MUC1 in one of the 11 controls. None of the control group were positive for both CK7 and MUC1, in contrast to eight of the nine patients with advanced breast carcinoma who were positive for both markers. The RT-PCR method thus appears sufficiently sensitive to identify circulating tumour cells in peripheral blood samples from patients with advanced breast carcinoma. However a high proportion of false-positive results was seen in the control population. More extensive investigation is required before the technique is likely to be of benefit clinically.

Perceptual responses and blood lactate concentration: effect of training state.

The present study investigated the effect of training state on ratings of perceived exertion obtained at the lactate threshold (LT) and fixed blood lactate concentrations (FBLC) of 2.0, 2.5, and 4.0 mM. Runners (N = 20) and nonrunners (N = 29) completed a progressive horizontal treadmill (TM) running test which allowed identification of the TM velocities associated with the LT and FBLC. Runners attained significantly higher TM velocities, greater VO2, greater VE, greater heart rate, and a lower ventilatory equivalent for oxygen (VE/VO2) at each exercise intensity, with the exceptions of heart rate at 4.0 mM and VE/VO2 at the LT. Compared to nonrunners, runners also attained higher VO2, VE, and heart rate relative to peak values at LT and 2.0, 2.5, and 4.0 mM. Despite these relative and absolute physiological differences, there were no differences between groups in local, central, or overall ratings of perceived exertion (RPE) (Borg scale) at any condition. The data from both groups were combined to give the following means and SD for overall RPE during horizontal running: at the LT-11.0 +/- 2.0, and at FBLC of 2.0 mM-13.7 +/- 2.1, 2.5 mM-14.5 +/- 1.8, and 4.0 mM-16.5 +/- 2.3.

Effect of exercise modality on ratings of perceived exertion at various lactate concentrations.

The effect of exercise modality on the relationship between ratings of perceived exertion (RPE), blood lactate concentration, oxygen uptake (VO2), and heart rate (HR) was examined in 29 untrained male subjects who completed counterbalanced VO2max/lactate threshold (LT) protocols on a cycle ergometer (CE) and treadmill (TM). Heart rate, VO2, and RPE were determined at power outputs corresponding to LT and fixed blood lactate concentrations (FBLC) of 2.0, 2.5, and 4.0 mM and during maximal exercise. A repeated measures ANOVA indicated that, despite significant differences across exercise modality in HR and VO2 at LT, FBLC, and maximal exercise, no significant differences in RPE were found between exercise modalities during leg exercise. Mean (+/- SD) respective values for overall RPE at LT and FBLC of 2.0 mM, 2.5 mM, 4.0 mM, and max were 10.2 (2.2), 13.1 (2.1), 14.1 (2.3), 15.9 (2.3), and 18.8 (1.3) for the CE and 10.8 (1.9), 13.8 (1.8), 14.6 (1.6), 16.2 (2.6), and 18.5 (1.5) for the TM. It was concluded that exercise modality does not affect the perception of exertion at LT, FBLC, or maximal exercise and that a strong relationship exists between RPE and blood lactate concentrations.

Nonoperative treatment of ulnar collateral ligament injuries in throwing athletes.

Ulnar collateral ligament injury of the elbow in throwing athletes is a common occurrence, and either operative or nonoperative treatment is an option. The results of operative repairs and reconstructions have been well documented in the literature; however, little information has been reported on the outcome of nonoperative treatment. From 1994 to 1997, we evaluated 31 throwing athletes with ulnar collateral ligament injuries. The purpose of this study was to determine what percentage of athletes could return to their sport without surgical intervention and to identify factors that would predict return to full competition by an athlete treated nonoperatively. The factors studied included acute versus insidious onset of symptoms, the duration of symptoms before treatment, and age. Nonoperative treatment, which included a minimum of 3 months' rest with rehabilitation exercises, allowed 42% of the athletes in our study (N = 13) to return to their previous level of competition. Those who did return did so at an average of 24.5 weeks after diagnosis. No predictive findings obtained either through the patient's history or physical examination were found that would assist the clinician or athlete in predicting the success of nonoperative treatment.